1. Field of the Invention
The present invention relates generally to the fields of gene therapy and transplantation. More specifically, the present invention relates to an adenoviral vector encoding an anti-apoptotic Bcl-2 gene for cytoprotection, gene therapy, and cellular and organ transplantation.
2. Description of the Related Art
The delineation of the molecular basis of cancer allows for the possibility of specific intervention at the molecular level for therapeutic purposes. To an increasing extent, the genetic lesions associated with malignant transformation and progression are being identified [1-3]. Thus, not only in the context of inherited genetic diseases, but also for many acquired disorders, characteristic errors in patterns of gene expression may be precisely defined. Based on this understanding, the rationale to develop novel therapeutic modalities on gene therapy approaches has become increasingly compelling.
The elucidation of the genetic basis of inherited and acquired diseases have rendered gene therapy both a novel and rational approach for these disorders [4-14]. To this end, three main strategies have been developed: mutation compensation, molecular chemotherapy, and genetic immunopotentiation. For mutation compensation, gene therapy techniques are designed to rectify the molecular lesions in the cancer cell etiologic of malignant transformation. For molecular chemotherapy, methods have been developed to achieve selective delivery or expression of a toxin gene in cancer cells to achieve their eradication. Genetic immunopotentiation strategies attempt to achieve active immunization against tumor-associated antigens by gene transfer methodologies. For these conceptual approaches, human clinical protocols have entered Phase I clinical trials to assess dose escalation, safety, and toxicity issues.
Two general approaches have been employed. Firstly, the ex vivo approach, whereby target cells are genetically modified extracorporally, followed by reimplantation. This strategy capitalizes on the fact that the current generation of vectors can achieve efficient gene transfer in vitro, as well as in the fact that a level of biosafety characterization is achievable. Despite the fact that this strategy has been employed successfully in a number of animal model systems to achieve genetic correction, the translation into human clinical trials has yielded only a relatively few parenchymal cells that were manipulated. Whereas these methods have been of utility for selected disease contexts, for many disease states this approach does not offer a practical means to achieve a meaningful genetic intervention. Based on this concept, a second approach for gene therapy has been developed based on direct, in vivo delivery of therapeutic genes to target parenchymal cells in situ within their organ context.
For direct in vivo gene transfer, a variety of vector systems have been evaluated in a loco-regional context. These include viral vectors, such as adenovirus, retrovirus, herpes virus vectors, and adeno-associated virus (AAV). In addition, nonviral vectors have been employed to this end, including liposomes, molecular conjugates, polymers and other vectors. In general, despite their utilities, these vectors have been limited in their ability to accomplish highly efficient in vivo gene delivery to relevant target organs. In this regard, the development of strategies to target airway epithelium for cystic fibrosis (CF) gene therapy has allowed the analysis of the gene transfer dynamics of a variety of vector approaches in this context [15-20]. In these studies, a low level of in vitro gene transfer was noted with both types of vectors. However, recombinant adenoviral vectors achieved the highest levels of in situ gene transfer to airway epithelium [21-25].
Based on these results, human clinical trials were developed employing adenoviral vectors [26]. Further, for metabolic and blood disorders, such as hemophilia, in vivo gene therapy approaches have been developed largely via in vivo gene transfer to the liver [27-28]. Here again, the highest levels of transduction of hepatocyte parenchymal cells after in vivo gene delivery have been observed with adenoviral vectors [29-31]. Thus, adenoviral vectors have shown the highest efficiency of in vivo gene delivery in the context of in situ transduction to a variety of parenchymal cells [32].
There are additional advantages to using adenoviral vectors. First, the adenovirus has been extensively studied and is the most widely employed gene therapy vector. Secondly, adenovirus has been produced consistently with high titers. Thirdly, with the use of new recombinant technologies the risk for replication-competent adenovirus in the preparations is low [33]. Fourthly, previous limitations concerning the size of therapeutic gene inserts into the adenovirus genome have been superceded with the development of recombinant adenovirus deleted in multiple early and late genes. Fifthly, recent evidence has shown that inducible systems can be developed in the adenovirus context such as the tetracycline on/off and Cre/Loxp systems [34-35]. Finally, the delivery and/or expression of adenoviral-encoded transgenes can be targeted to the appropriate set of cells by a variety of mechanisms, including modifications of the binding properties of the adenovirus as well as regulation of expression of transgenes by tissue or tumor specific promoters [36].
Loss of adenoviral transfected cells rather than transgene extinction has been suggested as an important factor responsible for limited in vivo expression of therapeutic genes. Cytoprotection of the viral-vector host cells against the immune system and the cytotoxic effects of the viral products may allow significant prolongation of the transgene expression. Cells and organs which express Bcl-2 are more resistant to cytotoxic effects of T lymphocytes, cytokines, free oxygen radicals and other mediators of the immune system.
Programmed cell death or apoptosis plays an important role in a wide variety of physiological processes, including for example removal of redundant cells during development, elimination of autoreactive lymphocytes, and eradication of older, differentiated cells in most adult tissues with self-renewal capacity. Disregulation of this physiological mechanism for cell death has been implicated in a variety of human diseases, ranging from cancer to autoimmunity, where insufficient cell death can figure prominently, to AIDS and neurodegenerative disorders, where excessive death of T-lymphocytes and neurons occurs as well as in acute diseases such as infection, ischemia-reperfusion damage and infarction. The process of cell death and its morphological equivalent xe2x80x9capoptosisxe2x80x9d can be subdivided into three different phases: initiation, effector and degradation. Whereas the initiation stage depends on the type of apoptosis-inducing stimulus (grow factor deprivation, cytokines, thermal/mechanical injury, irradiation, etc.) the effector (which is still subject to regulation) and degradation (beyond regulation) stages are common to all apoptotic processes.
The family of bcl-2 related proteins constitutes one of the most biologically relevant classes of apoptosis-regulatory gene products acting at the effector stage of apoptosis. Several biological effects of bcl-2 on intact cells have been reported. Bcl-2 might act on plasma membrane (prevention of phosphatidylserine), on the cellular redox potential (decrease lipid peroxidation, inhibition of reactive oxygen species, increase in catalase and superoxide dismutase, elevated NAD-/NADH ratio), effects on proteases (inhibition of caspase 3 and 6). Other effects of bcl-2 include effects on intracellular ions (prevention of cytoplasmic acidification, inhibition of Ca++ uptake into the nucleus) and effects on mitochondria (inhibition of pre-apoptotic mitochondrial transmembrane potential disruption, prevention of Ca++ influx, prevention of cytochrome C outflow from the intermembrane space, etc.). One of the first common manifestations of the apoptotic process, irrespective of the cell type and the induction stimulus, is a disruption of the mitochondrial membrane function that marks the xe2x80x9cpoint of no returnxe2x80x9d of the apoptotic process. Given the functional importance of bcl-2 gene in apoptosis control, it constitutes prime targets for therapeutic interventions on numerous disease states.
Islet transplantation is associated with high incidence of primary non-function. Organ damage has been demonstrated since organ procurement, preservation and reperfusion. In the case of the pancreatic islets, additional xe2x80x9cinsultingxe2x80x9d factors like the islet isolation, culture, poor vascularization and proinflammatory cytokine release after transplantation make the islet transplantation a high risk procedure for primary non-function. In normal physiology the islets are vascularized by an afferent arteriole (arterial blood) and are innervated by cholinergic adrenergic and peptidenergic innervations. In a period of weeks after transplantation, the islets are not normally vascularized. In the case of intraportal administration, immediately after transplantation, islets are vascularized by only portal venous blood and after a period of several weeks are presumably vascularized by hepatic arterial blood. The changes in the vascular and neurophysiological environments may be coincident with the complete normal islet function. Poor islet vascularity early after transplantation has been determined as an important cause of islet dysfunction and survival after transplantation. Infiltration of the pancreatic islets by immune/inflammatory cells (insulitis), followed by loss of the insulin producing beta cells is the characteristic histological feature of insulin-dependent diabetes mellitis (IDDM). Islet cell destruction may involve direct contacts between the infiltrating monocytic/lymphocytic cells and islet beta cells and/or may result from the release of soluble mediators from the effector immune cells. Cytokines produced by activated cells infiltrating islets are candidate molecules mediating impaired function and destruction of beta cells in IDDM. Products of inflammation, such as Interleukin-1xcex2 and nitric oxide, has been shown to impair early function of pancreatic islets after intrahepatic transplantation. Rabinovitch et al has reported DNA fragmentation as an early event in cytokine induced beta cell destruction.
Liver graft function after transplantation is determined largely by the quality of the donor organ at harvest and the secondary damage during the hypothermic preservation time, warm ischemia and reperfusion injury. These processes may also play a role in the incidence of rejection, vascular and biliary complications, and overall graft and patient survival. Experimental evidence suggested that ischemic injury involves a loss of mitochondrial respiration and consequently ATP depletion. Although cell necrosis is the assumed end-result of preservation and ischemia-reperfusion injury, programmed cell death (apoptosis) results from these identified mechanisms of ischemic injury and has been demonstrated during preservation-induced damage to cultured endothelial cells, liver preservation, and is a frequent event in post-reperfusion biopsy specimens of human liver grafts. Apoptosis has been observed mainly in hepatocytes and sinusoidal lining cells and its intensity correlates with postoperative signs of hepatocyte injury. Genetic modification of liver grafts with the proposed recombinant adenovirus vector might allow the use of xe2x80x9cmarginal liver graftsxe2x80x9d, such as livers from old donors, fatty livers, etc.
The significance of ischemia-reperfusion injury in organ transplantation is clearly an important determinant of early and late graft dysfunction. The mechanism and mediators involved in the ischemia/reperfusion injury of liver allografts remain unknown, various etiologic factors have been identified, these include activation of proteases and phospholipases, alteration in calcium concentrations, ATP depletion, cell damage by free radicals, cytokines, chemokines and edothelins, inhibition of nitric oxide synthesis and an active role of cell of the immune system, such as neutrophils and kuppfer cells. Apoptosis is a frequent event in post-reperfusion biopsy specimens of human liver grafts, its main cellular target are hepatocytes and sinusoidal lining cells and its intensity correlates with postoperative signs of hepatocyte injury.
Apoptosis of endothelial cells has been described during organ preservation for transplantation. Cytoprotection of endothelial cells represent an attractive alternative for improved organ preservation. Apoptosis also plays an important role in vascular disease like atherosclerosis, therefore, cytoprotection of endothelial cells might prevent the development of atherosclerotic changes secondary to multiple stimuli, including chronic rejection in clinical transplantation.
The prior art is deficient in the lack of an adenoviral vector encoding a functional anti-apoptotic Bcl-2 gene. Further, the prior art is deficient in the lack of effective means of genetic cytoprotection using an adenoviral vector encoding an anti-apoptotic Bcl-2 gene. The present invention fulfills this long-standing need and desire in the art.
The present invention is the first description of an adenoviral vector type 5 (E1 deleted) construct capable of producing a functional human Bcl-2 molecule. This approach has applicability to protect adenoviral vector transduced cells, to improve organ preservation for transplantation, to protect from ischemia/reperfusion injury, to protect cells after cellular transplantation, or organs for both allotransplantation and xenotransplantation, and to protect endothelial cells from various inducers of injury.
In one embodiment of the present invention, there is provided an adenoviral vector encoding an anti-apoptotic Bcl-2 gene. Preferably, the adenoviral vector is type 5 with E1 deleted and contains an expression cassette with the Bcl-2 gene under the control of a cytomegalovirus promoter. Preferably, the Bcl-2 gene is human Bcl-2 gene.
In another embodiment of the present invention, there are provided various applications of the disclosed adenovirus vector, including reducing ischemia/reperfusion injury to the liver, improving organ preservation for transplantation, cytoprotecting endothelial cells during cold preservation, cytoprotecting pancreatic islets, and enhancing/prolonging expression of a transgene in a cell.
In still another embodiment of the present invention, there is provided a pharmaceutical composition, comprising the disclosed adenoviral vector and a pharmaceutically acceptable carrier.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.